Maintenance of genomic integrity is paramount to cell survival. Precise duplication of our genome and appropriate cellular response to genotoxic stress is critical to maintain genome stability. Origin Recognition Complex (ORC, composed of six subunits) and ORC-Associated (ORCA) are required for the initiation of DNA replication and regulate heterochromatin organization. Multiple subcomplexes of ORC and/or individual ORC subunits regulate different aspects of cell cycle progression and thus play pivotal roles in the maintenance of genomic stability. The long-term goal of my laboratory is to understand how ORC executes and coordinates various aspects of cell growth, proliferation and survival. The smallest subunit of ORC, Orc6, is required for DNA replication and also coordinates cytokinesis. The scientific premise of this proposal that human Orc6 is required for DNA replication progression and DNA Damage Response is based on strong preliminary data. We have exciting preliminary data demonstrating yet another novel role of Orc6, in DDR. We observe that the levels of Orc6 increase upon DNA damage and that Orc6 undergoes specific phosphorylation during oxidative DNA damage. Further, cells lacking Orc6 fail to activate ATR upon DNA damage. The objective of the present proposal is to answer fundamental questions on the roles of Orc6 in regulating S-phase and during DDR. Our hypothesis, based on preliminary data, is that Orc6 interacts with replication fork components and facilitates DNA replication and upon DNA damage the loss of this interaction inhibits replication fork progression. With our expertise and experience in cell biological and biochemical characterization of Orc6 and strong preliminary data, we are ideally positioned to pursue the following specific aims: 1) Determine the role of Orc6 in replication fork progression. 2) Determine the role of Orc6 in DNA damage response. 3) Determine the role of post-translational modifications on Orc6 in DDR and cell cycle progression. An important question in the field that has remained to be answered is why do ORC proteins remain bound to chromatin in postG1 cells? This proposal is significant because we address this question by studying how Orc6 regulates replication fork progression and it evaluates for the first time the role of preRC proteins in DNA damage response. This proposal is conceptually innovative because we will rigorously dissect novel regulatory mechanisms of Orc6 in DNA replication progression and DNA damage response. This proposal is technologically innovative because it employs state of the art cell biological techniques, including super-resolution imaging combined with biochemical and single molecule biophysical approaches. Understanding how Orc6 governs multiple pathways including DNA replication, mitosis and DDR is expected to uncover novel pathways that would be useful to prevent tumorigenesis and key to allow more effective therapeutic targeting to combat cancer.